Methods and apparatus/instrumentation for detecting, capturing and isolating rare circulating cells, include, but are not limited to, circulating tumor cells (CTCs) and circulating stem cells (CSCs) from preparations of clinical samples collected from a subject. These methods allow rapid detection, capture and isolation of cells of interest that are identified using a rare cell detection system. The methods may employ the labelling of affinity reagents, such as antibodies, for immunoassays with removable signal molecules to allow serial analyses with multiple probes coupled to signalling molecules. The rare cell detection methods allow qualitative and quantitative immunoanalysis, gentle removal of the signal molecules and then re-analysis of the sample using affinity reagents toward other targets. The samples can be analyzed multiple times in the same manner. The captured cells can then be used for down-stream analyses such as, but not limited to, genetic and proteomic assessments.
Circulating tumor cells in the blood stream play a critical role in establishing metastases. The clinical value of CTCs as a biomarker for early cancer detection, diagnosis, prognosis, prediction, stratification, and pharmacodynamics have been widely explored in recent years. However, the clinical utility of current CTC tests is limited mainly due to methodological constraints. There is a need for methods, reagents and devices for detecting increased metabolic activity of cancer cells for rapid detection of the cancer cells without the need to enrich a sample.
Rare circulating cells such as circulating tumor cells (CTC) and circulating stem cells (CSC) are generally thought to represent untapped opportunities for diagnosing and monitoring pathologies. In the case of CTCs/CSCs, the cells are assumed to be shed from primary or secondary tumors of patients with advanced cancer and have been detected in the peripheral blood of patients with advanced stages of most types of solid tumor cancers. However, CTCs have also been detected in patients with localized cancers, which may be indicative of increased risk of progression to metastatic disease or very early tumor development. It is possible the rapidly growing pre-malignant lesions shed epithelial cells in sufficient quantity to be captured from the peripheral blood and analyzed for early diagnosis.
Since CTCs are mainly characterized and identified by their morphology and immunostaining pattern, their heterogeneity is a major obstacle for CTC detection. The CTCs derived from different types of tissues significantly distinguish from each other with different size, shape, and immunophenotyping profile. However, there is broad morphological and immunophenotypical variation within CTCs derived from the same tissue of origin. During epithelial to mesenchymal transition, the expression of epithelial markers on CTCs, such as epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK), may be down-regulated and become undetectable.
Therefore, accurate detection of CTCs based on morphological and immunophenotypical profiling is still challenged. Additionally, CTCs may be damaged and fragmented, in vivo and/or in vitro, due to multi-step cell preparation processes, causing inaccurate detection and misinterpretation.
CTCs are characterized as non-leukocytic, nucleated cells that are typically epithelial in origin, and maintain significantly larger diameters than normal blood cells. However, the morphological features of CTCs are now known to be less clearly defined. It is accepted that a significant number of CTCs may lose their epithelial markers and express the phenotypic markers of epithelial-mesenchymal transition (EMT). Subsets of CTCs may represent viable metastatic precursor cells capable of initiating a metastatic lesion. Molecular and phenotypical differences between CTCs and the primary tumor have been documented and may vary by cancer type and disease progression. Additionally, it has been demonstrated that there is heterogeneity among a patient's CTCs. These complexities introduce additional challenges for interpreting CTC analysis results. The analytical methods/assays used will be critical to establishing a common set of criteria describing CTCs.
From a technical standpoint, almost all CTC assays have three major steps: 1) blood sample preparation and tumor cell separation; 2) cell staining by antibodies or gene probing by DNA probes; and 3) CTC detection. A platform that can characterize the oncogenic alterations in the CTCs may aid in identifying therapeutic sensitivity/resistance which would be critical for early modification of therapeutic regimens contributing to more effective personalized health care. It has recently been suggested that clusters of CTCs may be relatively protected from cell death and that the presence of clusters may be a better marker of metastatic potential than single CTCs. Current enrichment methodologies are likely to disrupt CTC clusters thereby missing these potential indicators of metastatic potential. These enrichment protocols result in a biased capture of the CTCs detecting only those CTCs that conform to the predetermined criteria for capture. Thus, methods that overcome the limitations of current techniques of biased enrichment and disruption of CTC clusters are critical to realize the full potential for CTC detection and characterization to positively impact patient outcome. Additionally, methods for cell capture and isolation are needed if the cells of interest are to be used for downstream analyses. The methods described herein are designed to capture the cells subsequent to detection and characterization.
Current immunostaining techniques employ affinity reagents such as, but not limited to, antibodies, antibody fragments and engineered binding molecules coupled to a readout molecule such as a fluorescent molecule or an enzyme that generates luminescence or a chromophore. Stripping the antibodies from an immunoblot is a common practice but requires harsh conditions such as 2% SDS at 50° C. or buffers with an acidic pH of 2. A clinical sample on a microscope slide will not withstand these harsh conditions and equally effective stripping conditions have not been developed. Photobleaching a fluorescent signal molecule is not sufficiently efficient to abolish the signal. Thus, currently, there is a need for a method for immunostaining a tissue section or cells attached to a microscope slide more than one time. Immunofluorescence microscopy allows for multiple antibodies to be used in a single immunostaining procedure.
Generally, the number of fluorophores that can be used in a single sample has been limited to 5 due to the necessity for using fluorophores with distinct absorbance and emission spectra.
For quantitation of antigens in a single immunostain procedure, the user is limited to emitters of blue, green, orange, red and near infra-red wavelengths to assure excitation/emission signals that do not overlap. However, the ability for quantitation of only 5 antigens limits the information that can be gathered from a cell of interest. The ability to remove the signal from a previous immunostaining procedure and stain the sample using antibodies to additional proteins of interest would greatly enhance the utility of immunostaining for cell characterization.
The fluorescent material may be any suitable fluorescent marker dye or any other suitable material which will identify the cells of interest. A smear treated in this manner, which may include the blood and/or components of the blood, is prepared and optically analyzed to identify rare cells of the targeted type. For statistical accuracy it is important to obtain as large a number of cells as required for a particular process, in some studies at least ten rare cells should be identified, requiring a sampling of at least ten million cells, and up to fifty million or more, for a one-in-one-million rare cell concentration. Such a blood smear typically occupies an area of about 100 cm2. It is to be understood, however, that this is simply one example and other numbers of cells may be required for statistical accuracy for a particular test or study. Other cell identifiers which are being used and investigated are quantum dots and nanoparticle probes. Also, while a rare cell is mentioned as a one-in-one-million cell concentration, this is not intended to be limiting and is only given as an example of the rarity of the cells being sought. The concepts discussed herein are to be understood to be useful in higher or lower levels of cell concentration.
There is a need for a reliable method that allows for easy removal of a readout molecule or label that will allow samples to be immunostained multiple times so that it may be processed for affinity-detection of additional proteins not targeted in a previous cycle. Such a method will allow for determining the proteomic profile of single cell of interest or lysates can be assessed using multiple antibodies.
Cellular transformation is associated with the reprogramming of cellular pathways that control proliferation, survival, and metabolism. Among the metabolic changes exhibited by tumor cells is an increase in glucose, fructose, galactose and amino acid metabolism. Despite the presence of sufficient levels of oxygen, tumor cells exhibit high levels of glycolysis. This observation is now exploited in the clinic for diagnostic purposes. Positron emission tomography (PET scan) using 2-deoxy-2(18F)-fluoro-D-glucose (18F-FDG), a glucose analogue, demonstrates a significant increase in glucose uptake in tumors compared with adjacent normal tissue. 18F-FDG, has become a routine clinical test for staging and restaging of malignant lymphoma and solid tumors. FDG is taken up by the same membrane transporters that take up glucose and is phosphorylated by the same hexokinases as is glucose. The difference is that when FDG is phosphorylated to become FDG-6-phosphate in the cell, it is metabolically trapped. It cannot go on to be stored as glycogen or go on to glycolysis the way glucose can; it is a polar molecule that cannot readily pass through the cell membrane to redistribute out of the cell. Thus, sites of active tumor will show up as foci of hypermetabolism, or “hot spots” on the subsequent PET scan images.
Recent studies indicate that the activation of proto-oncogenes, signaling pathways, and transcription factors, as well as the inactivation of tumor suppressors, induce the increased metabolic activity in cancer cells. Because tumor cells have increased metabolic activity relative to normal cells, rare, highly metabolic circulating tumor cells may be distinguished from the background of millions of non-transformed lymphocytes in a patient's blood sample using fluorescent glucose, glucosamine analogs, amino acids or stains that distinguish cells with high metabolic activity.
The advantage of using glucosamine analogs and amino acids is the availability of the amine group for conjugating any of a number of fluorophores. Thus, a fluorophore with the appropriate emission spectrum for use with the instrument can be used to generate the fluorescent glucosamine. The increased uptake of fluorescent glucosamine or amino acid by the circulating tumor cells relative to that of the normal white blood cell in the sample should provide a clear fluorescent disparity between the cells allowing for identification of the tumor cells. The fluorescent glucosamine or amino acid reagent will be used on live cells. It would be advantageous to distinguish tumor cells while maintaining viability. Maintaining cell viability increases the options for downstream analyses such as mRNA studies and culture of the tumor cells for proteomic analyses.
Moreover, several cell detection methods and apparatus have been proposed to detect rare cells. These include various types of automated microscopic imaging; immunomagnetic cell enrichment in combination with digital microscopy; use of reverse transcriptase polymerase chain reaction (RT-PCR) with some immunomagnetic isolation; fluorescence image analyses; fluorescence in situ hybridization (FISH); cell detection is flow cytometry (FC); laser scanning cytometry (LSC); use of a fiber optic bundle arranged to define an input aperture for viewing a sample on the translation stage; etc.
Conventional cell detection systems are complex, time consuming, and/or expensive. What is needed is a cell detection system that improves speed, reliability, and/or processing costs. Indeed, the purportedly fastest cell detection systems available on the market requires several hours to complete CTC detection for a single slide. There are also challenges in practice to make sure that cells bunching or clumping together does not prevent identifying each cell individually. Most current CTC detection technologies are based on enrichment of CTCs or removal of the white blood cells in a blood sample to be analyzed. The methods to achieve enrichment of CTCs are not sufficiently effective to allow confidence that all classes of CTCs are detected. Of the few technologies that do not rely on enrichment, the extended time required for analysis limits the utility of the technologies.
Accordingly, there is a need to address the aforementioned and other problems currently associated with rare cell detection. These include, without limitation, more specific markers and labelling strategies for detecting rare cells well as enhancing the throughput, sensitivity, and analytic functionality of current methodologies, systems, platforms and/or devices.